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Biochemical Systematics and Ecology, VoL 24, No. 1, pp. 1-12,1996 Copyright © 1996 Elsevier Science Ltd

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Defense Metabolites from the Marine Sponge Verongia aerophoba

BEATE WEISS,* RAINER EBEL,* MALTE ELBRh, CHTER,t MARIANNA KIRCHNERt and PETER PROKSCH%

*Julius-von-Sachs-lnstitut for Biowissenschaften, Universit~t W(Jrzburg, Mittlerer Dallenbergweg 64, D-97082 Werzburg, Germany;

tBiologische Anstalt Helgoland, Wattenmeerstation Sylt, Hafenstra~e 43, D-25980 List/Sylt, Germany; tBiologische Anstalt Helgoland, Meeresstation Helgoland, Postfach 180, D-27498 Helgoland, Germany

Key Word Index--Verongia aerophoba; sponges; bacteria; microalgae; gastropods; natural products; chemical defense.

Abstract - -The marine sponge Verongia aerophoba (syn. Aplysina aerophoba) accumulates isofistularin-3 and aerophobin-2 as major brominated isoxazoline alkaloids. Following disrupture of the compartmentation (e.g. by wounding) both isofistularin-3 and aerophobin-2 are enzymatically converted into aeroplysinin-1 which in turn gives rise to a dienone. Aeroplysinin-1 and dienone were shown to exhibit pronounced biological activities in various bioassays with marine organisms (bacteria, algae and molluscs) whereas their biogenetic precursors isofistularin-3 and aerophobin-2 were either inactive or exhibited only marginal activity. In the agar plate diffusion assay, aeroplysinin-1 and dienone were antibiotically active against eight different Gram-positive or Gram-negative marine bacteria including Alteromonas, Moraxella and Vibrio spp. Towards the marine Photobacterium phosphoreum the ECsos of aeroplysinin-1 and dienone were 3.45 and 1.37 ~M, respectively. Both compounds inhibited also the growth of the marine microalgae Coscinodiscus wailesii and Prorocentrum minimum. Towards the former, the ECsos of aeroplysinin-1 and dienone were 5.6 and 27.9 pM, respectively. In addition to their growth inhibitory activity, aeroplysinin-1 and dienone were algicidal as evident by their damaging effects on the algal cellular membranes. The polyphagous marine gastropod Littorina littorea was repelled when exposed to either aeroplysinin-1 or dienone that were added to seawater. The ECso of the most active compound aeroplysinin-1 was observed at 0.1 mM. It is suggested that the enzymatically catalysed conversion of brominated metabolites in V. aerophoba represents a wound-induced defense mechanism hitherto unreported from the marine environment.

In t roduct ion Marine sponges from the family Verongidae are a rich source of brominated isoxazoline alkaloids such as isofistularin-3 (1) or aerophobin-2 (2) that are thought to be biogenetically derived from dibromotyrosine (Tymiak and Rinehart, 1981). The Mediterranean sponge Verongia aerophoba Schmidt (syn. Aplysina aerophoba) which is also found at the Canary islands accumulates isofistularin-3 (1) and aerophobin-2 (2) in substantial amounts (up to 10% of the dry wt.) (-I-eeyapant et al., 1993a). As shown recently (Teeyapant and Proksch, 1993; Teeyapant, 1994) both isofistularin-3 (1) as well as aerophobin-2 (2) are enzymatically converted into aeroplysinin-1 (3) (Fig. 1) following disrupture of the compartmentation (e.g. by wounding). Aeroplysinin-1 (3) is spontaneously transformed into the dienone (4) under the mild alkaline conditions of seawater (Fig. 1) (Teeyapant, 1994). Preliminary data idicate that the enzyme(s) responsible for the bioconversion of isofistularin-3 (1) or aerophobin-2 (2) are also present in other members of the Verongidae analysed whereas they are missing in sponges from outside this group (Ebel et al.,

§Author to whom correspondence should be addressed.

(Received 4 August 1995; accepted 23 October 1995)

B. WEISS ETAL

H3CO Br

B r ~ OH Br .o- o J-

OH . ~ 1 HO ar

Br OCH~

0

OCH3 B ~ Br

\°jo 3

o Br. Br

H2NOC J 'OH

4

FIG. 1. WOUND-INDUCED BIOTRANSFORMATION OF ISOFISTULARIN-3 (1) AND AEROPHOBIN-2 (2) INTO DEFENSE METABOLITES AEROPLYSININ-1 (3) AND DIENONE (4).

unpublished data). Hence, the capability to convert isoxazoline alkaloids into aeroplysinin-1 (3) appears to be more widespread within the Verongidae than previously thought and is perhaps specific for this family.

The described enzymatic conversion of isoxazoline alkaloids in V. aerophoba is of ecological interest since it results in a pronounced increase of the biological activities of the products (3 and 4) compared to their precursors (1 and 2) as shown in the present study using marine bacteria, algae and gastropods as test organisms. It is suggested that V. aerophoba represents an example for a wound-induced chemical

DEFENSE METABOLITES OF V. AEROPHOBA 3

defense that relies on an enzymatic conversion of inactive storage compounds into active defense metabolites. Similar defense mechanisms are known from higher terrestrial plants (e.g. liberation of HCN from cyanogenic glycosides) (Luckner, 1984) but were hitherto unreported from the marine environment.

Materials and Methods Isolation and purification of sponge constituents. Sponges were collected at Rovinj (Croatia) and directly frozen ( -20°C). For isolation of compounds 1 and 2 freeze dried material was ground and extracted with MeOH. For isolation of compounds 3 and 4 sponges were extracted with 50% aq. MeOH to allow for the enzymatically catalyzed biotransformation of 1 and 2 (Teeyapant and Proksch, 1993). Extracts were partitioned between ethyl acetate and H20. The ethyl acetate layers were evaporated to dryness, redissoIved in a mixture of CH2CI2/MeOH 9 : 1 v/v) and subjected to column chromatography on silica gel using the same solvent mixture as mobile phase. Fractions (20 ml) were monitored by TLC on pre- made silica gel plates (Merck, Darmstadt, Germany) using the same mobile phase. Plates were viewed under UV-light (254 mm). Final purification of compounds 1-4 was achieved by column chromatography on Sephadex LH-20 with MeOH or with mixtures of MeOH/CH2CI2 as eluent or by column chromatography on reversed phase (Cls) lobar columns (Merck) using mixtures of MeOH and H20 (e.g. 80:20 v/v) as eluent. Compounds were identified based on their NMR and mass spectra as previously described (Teeyapant et al., 1 993b).

Antibacterial activity. For the agar plate diffusion assay marine bacteria obtained from the National Collections of Industrial and Marine Bacteria (NCIMB, Aberdeen, U,K.) and from the Biologische Anstalt Helgoland were used. Microorganisms were grown on a standard marine growth medium according to ZoBell (1946). The agar plate diffusion assay was performed according to standard procedures (DIN 58940). Compounds were dissolved in Me2CO. Known amounts were pipetted on paper discs (diameter: 6 ram). Following evaporation of the solvent the discs were transferred to petri dishes inoculated with the bacteria. Plates were incubated in the dark at 18°C. Inhibition zones were measured after 48 h. Each experiment was performed in triplicate. Experiments with Photobacterium phosphoreum ( 'Microtox" assay) were performed in triplicate according to standard procedures (DIN 38412). Luminescence of the bacteria was measured 30 min after addition of the compounds. ECsos of these as well as of the following experiments were calculated by probit-analysis.

Experiments with microalgae. Coscinodiscus wailesii G ran et Angst (Bacillariophyceae) and Prorocentrum minimum (Pav,) Schiller (Dinophyceae) were from a laboratory culture grown in F/2 medium according to Yates et aL (1 973). Algae used for the growth inhibitory experiments came from cultures that were in the exponential growth phase, Compounds were dissolved in known amounts of DMSO and added to the cultures (final concentration of DMSO in the medium: 0.2%). Controls were treated with the carrier only. Treated algae and control algae were cultivated at 18°C under a light/dark rhythm (12:12 h). Cell numbers of treated cultures and of controls were counted every 24 h. Experiments were performed in triplicate.

Experiments with gastropods. Fully grown individuals of the marine gastropod Littorina littorea (L.) collected at Helgoland (Germany) were used for the experiments. Specimens of L, littorea that were collected at low tide were placed in jars with fresh seawater 30 min prior to the experiments. For the experiments known amounts of compounds 2-4 were dissolved in 0.1 ml DMSO and added to jars with 20 ml of fresh seawater. Snails were then put individually in the jars (10 replications for each concentration analysed). Controls were placed in jars containing only seawater and DMSO. The number of snails that had withdrawn into their protective shells as a response to the added compounds was counted after 30, 60 and 180 min.

Results Antibacterial activity Compounds 1 -4 were screened for anti-bacterial activity against eight different strains of marine bacteria using the agar plate diffusion assay. Aeroplysinin-1 (3) as well as dienone (4) inhibited the growth of all marine bacteria when tested at a dose of 100 i~g/disc (Table 1). Both compounds were most active against Vibrio sp., V. anguillarum, Cytophaga/Flexibacter sp. and Moraxella sp. Towards several of the analysed bacteria the antibiotic activities of aeroplysinin-1 (3) and dienone (4) extended even to doses as low as 5 tlg/disc (Table 1). Aerophobin-2 (2) and isofistularin- 3 (1) proved to be inactive against all strains of bacteria studied (Table 1).

Dose-response studies were conducted with the marine luminescent Photobacterium phosphoreurn which is a well established test organism for monitoring toxins in seawater (Krebs, 1983). Intensity of light emitted from P.

4. B. WEISS ETAL.

phosphoreum in the presence of aeroplysinin-1 (3), dienone (4), aerophobin-2 (2) or isofistularin-3 (1) was used to determine the antibiotic activity of the respective compounds. From the dose--response curves obtained (Fig. 2), the ECsos of aeroplysinin-1 (3) and dienone (4) were calculated as 3.45 and 1.37 #M (corresponding to 1.16 and 0.44 t~g ml .1), respectively Aerophobin-2 (2) and isofistularin-3 (1) had a slight effect on light emittance of R phosphoreum only when tested at concentrations of 100 t~M each (Fig. 2).

A/gicida/ and growth inhibitory activity Growth inhibitory and algicidal activities of aeroplysinin-1 (3), dienone (4) and

aerophobin-2 (2) were studied using the marine microalgae Coscinodiscus wai/esi/ and Prorocentrum minimum. Isofistularin-3 (1) was not included in these bioassays due to the limited amounts of compound available. Compounds 2 - 4 were added to the liquid growth media of C. wai/esii and R minimum at a final concentration of

1ABLE 1. ANTIBIOTIC ACTIVITY (agar plate diffusion assay) OF AEROPLYSININ-1 (3), DIENONE (4), AEROPHOBIN-2 (2)

AND ISOFISTIJLARIN 3 (1) AGAINST SEVERAL MARINE BACTERIA

Diameter of inhibitory zone (ram)

Compound t 2 3 4 Dose (pg/disc) 100 100 100 50 5 100 50

Bacterium A/teromonas spec.' NCIMB 224 11 7 10 9

Cytophaga/Flexibacterspec.*, NCIMB 251 30 n.d nd 28 n d n.d, Moraxella spec," NCIMB 308 22 20 19 16 7

Pseudornonas fluorescens" N C I M B 129 8 9 7 7

SerraUa p/ymuthzca" t 1 8 14 11 7

Vibrio spec" 28 24 1 2 24 21 8 Vibrio angu#/arum" NCIMB 407 27 24 11 24 21 7

P/anococcus citreust NCIMB 1495 14 n.d, n d 14 n.d. n.d.

Numbers of compounds refer to Fig 1; n.d =antibiotic activity not determined. • G ram- negative bactena tGram positive bacteria

t 00% . . . . . . . . . . . . . . ~ =

e- 6C)% ~ "~-'-----6.__.

t--

E 4 0 %

J

2 0 %

- I I - Controls, Isofistularin-3, Aerophobin-2

4~- Aeroplysinin-1

-~-Dienone

0%

o o 2 o 40 6 0 8o lOO

Concentration [pM]

FIG, 2 ANTIBIOTIC ACTIVITY OF AEROPLYSININ 1 (3), DIENONE (4), AEROPHOBIN 2 (3) AND ISOFISTULARIN-3 (1) TOWARDS PHOTOBACTERIUM PHOSPHQREUM AS INDICATED BY ITS LUMINESCENCE IN THE PRESENCE OF THE RESPECTIVE COMPOUNDS. Luminescence of Ft phosphoreum was measured 30 rain after addition of the compounds


8 mg 1-1 each (corresponding to 24 I~M for aeroplysinin-1 and dienone and 16 #M for aerophobin-2). Growth of C. wailesiiwas slowed by approximately 50% compared to controls in the presence of dienone (4) and was completely stopped in the presence of aeroplysinin-1 (3) (Fig. 3).When tested against R minimum aeroplysinin-1 (3) and dienone (4) were similar in activity causing complete growth inhibition (Fig, 4). Aerophobin-2 (2) in comparison had no or only a slight inhibitory effect on the growth of C. wailesii or R minimum (Figs 3 and 4).

Coscinodiscus wailesii was chosen for dose-response experiments with aeroplysinin-1 (3) and dienone (4). Both compounds were added to the growth media at concentrations ranging from 1 to 6 mg 1-1 (3-18 I~M). From the dose- response curves (data not shown), the ECsos of aeroplysinin-1 (3) and dienone (4) were calculated as1.9 mg1-1 (5.6 #M) and 9.0 mg 1-1 (27.9 IIM), respectively (Table 2).

In addition to their growth inhibitory effects both aeroplysinin-1 (3) and dienone (4) showed algicidal activity. When viewed under the light microscope the cytoplasm from cells of C. wailesii appeared severely damaged. Cell contents were either shrunk resembling plasmolytic cells or were protruding indicating damage of the cellular membranes (Fig. 5). Similar effects were observed for cells of R minimum when grown in the presence of either aeroplysinin-1 (3) or dienone (4) (data not shown).

Repellency towards the gastropod Littorina littorea Repellency of aeroplysinin-1 (3), dienone (4) and aerophobin-2 (2) was studied

using the polyphagous marine gastropod Littorina fittorea as test organism.

250

200

~ - - 150 x

E c

0 100

50 ¸

Control

-# - Dienone

Aeroplysinin- 1

-e - Aerophobin-2

0 4 ~

0 24 48 72 96 120

Time [h ]

FIG. 3. GROWTH (measured as time dependent increase of cell numbers) OF COSClNODISCUS WAILESII IN LIQUID GROWTH MEDIUM SPIKED WITH AEROPLYSININ-1 (3), DIENONE (4) OR AEROPHOBIN-2 (2) AT A FINAL CONCENTRATION OF8mg l 1 EACH.

6 B. WEISS ETAL.

300

250

200 m x

~ Q

~ 150 E

0

I O 0

-IF- Control

-~- Dienone

-~ - Aeroplysinin-1

-o- Aerophobin-2 i

S0,

I 0 - ~ - - - - 4 - - - - - - - - - ~ - - - - I I t t

0 24 48 72 96 120 144

T i m e [h)

FIG 4. GROWTH (measured as time dependent increase of cell numbers) OF PROROCENTRUM M I N I M U M IN LIQUID GROWTH MEDIUM SPIKED WITH AEROPLYSlNINq (3), DIENONE (4) OR AEROPHOBIN-2 (2) AT A FINAL CONCENTRATION OF8mg l ~ EACH

TABLE 2 ECsos OF AEROPLYSININ-1 (3) AND DIENONE (4) TOWARDS GROWING CELLS OF

COSCINODISCUS WAILESII

ECso in Compound mg I ~ ~LM

3 ~ 9 5.6 4 90 27.9

Compounds ;3 and 4 were added to cultures of C wailesii at a range of concentrations Cell

number was monitored after 120 h and compared to controls treated w i t h t h e carrier (DMSO) only, From the respective dose-response curves EC.~oS were calculated by probit a n a l y s i s

Isofistularin-3 (1) was not available in sufficient amounts to be included in this experiment. In the first experiment aeroplysinin-1 (3) was added to L. l i t torea kept in seawater at a series of concentrations ranging from 0.05 to 0.3 mM. Snails responded to increasing concentrations of aeroplysinin-1 (3) by withdrawal of the soft body into the protective shell. From the dose-response curve obtained (Fig. 6) the EC5o of aeroplysinin-1 (3) (after 30 min of exposure) was calculated as 0.1 raM.

FIG. 5. CELLS OF COSClNODISCUS WAILESll EXPOSED TO AEROPLYSlNIN-1 (3) (8 mg I 1 or 24 ~M) VIEWED UNDER THE LIGHT MICROSCOPE (10-fold magnification). Photograph was taken 48 h after exposure of cells to compound 3.


In the second experiment behavioral changes of snails exposed to aeroplysinin-1 (3), dienone (4) or aerophobin-2 (2) (0.2 mM each) were protocolled after 30, 60 and 180 rain (Fig. 7). At each observation aeroplysinin-1 (3) proved to be most active compound followed by dienone (4). Aerophobin-2 (2) in comparison elicited no or only weak (after 180 min of exposure) behavioral responses of L. littorea. Sensitivity of the snails to the compounds generally increased with exposure time which was most evident for dienone (4) (Fig. 7).

1 0

~6 u m

'~ 4 .IO

Z 2

0 I [

0 0.1 0.2 0.3

Concentration [raM]

FIG. 6. REPELLENCY OF AEROPLYSININ-1 (3) TOWARDS LITTORINA LITTOREA. The molluscs were kept in seawater spiked with increasing concentrations of compound 3. The number of specimens that had withdrawn into their protective shells (number of reacting snails) was counted after 30 rain.

1 0

t-

C u m

¢,j

E -,,1

Z

30 60 180

Time of exposure (rain)

[ ] Control

• Dienone

• Aeroplysinin-1

[ ] Aerophobin-2

FIG. 7. TIME-DEPENDENT INCREASE OF THE REPELLENT ACTIVITY OF AEROPLYSININ-1 (3) AND DIENONE (4) TOWARDS LITTORINA LITTOREA. After 30, 60 and 180 rain snails that had withdrawn into their protective shells (number of reacting snails) were counted. Compounds were tested at a concentration of 0.2 mM each. Controls were treated with the carrier (DMSO) only.

10 B. WEISS ETAL

None of the snails died during the experiment. When transferred to fresh seawater the snails became active again and searched for food indicating that aeroplysinin-1 (3) or dienone (4) in spite of their apparent repellent activity had no acute toxic effects on L. /ittorea.

Discussion Our study demonstrates antibiotic, growth inhibitory and repellent activities of aeroplysinin-1 (3) and dienone (4) towards marine bacteria, algae and gastropods and thus supports the defensive nature of these compounds that was suggested earlier (-I-eeyapant and Proksch, 1993).

Antibiotic activity of sponge metabolities has frequently been demonstrated in the past (Burkholder, 1973; Braekman and Daloze, 1986; McCaffrey eta/., 1985; Mebs, 1989). However, most of these studies were conducted with terrestrial rather than marine bacteria. Previously the antibiotic activity of aeroplysinin-1 (3) and dienone (4) was proven using terrestrial Gram-positive and Gram-negative bacteria (Teeyapant eta/., 1993b). These data were now confirmed using several marine bacteria such as A/teromonas, Moraxel/a or Vibrio spp (Table 1). The EC5os of compounds 3 and 4 towards P phosphoreum which is a well-established model for the analysis of water-borne toxins (Bulich eta/., 1990; Hambsch et a/., 1990; Lawton eta[ , 1990) (3.45 and 1.37 p M respectively, Fig. 2) are comparable to those for example of pentachlorphenol (ECso: 1.3 ~tM), lead (ECso: 1.9 pM) or cadmium ions (EC5o: 71 HM) (Krebs, 1983) which are known for their pronounced toxicity.

Aeroplysinin-1 (3) and dienone (4) are also active against microalgae. They inhibit the growth of C wai/esfiand R minimum (Figs 3 and 4) at micromolar concentrations (ECso of compound 3 towards C wai/esii: 5.6 pM) (Table 2) and show algicidal activity as evident by the severely damaged cells of C wailesfi (Fig. 5). This observed algicidal effect is probably due to damage of the cellular membranes since dienone (4) was previously shown to be haemolytic towards red blood cells (Teeyapant, 1994).

It is possible that the demonstrated antibacterial and growth inhibitory or algicidal activity of aeroplysinin-1 (3) and dienone (4) are involved in the suppression of fouling organisms Bacteria as well as microalgae (diatoms) are usually among the first organisms to settle on marine surfaces prior to attachment of macroalgae or epibiotic invertebrates (Bakus eta/ . 1986; Davis et a/., 1989; Melton and Bodnar, 1988). Natural products from invertebrates (e.g. eudistomins from the ascidian Eudistoma ofivaceum or amathamide alkaloids from the bryozoan Amathia wi/soni) have frequently been suggested (Davis, 1991; Walls et aL, 1991) to suppress the growth of fouling organisms and hence to be responsible for the remarkably clean surfaces of many marine invertebrates (such as V. aerophoba). The presence of the respective metabolites on the surface of the producing organism or continuous release into the seawater, however, which should be expected for any anti-fouling substance has rarely been described Recently it was shown that wounded specimens of // aerophoba release aeroplysinin-1 (3) into seawater (Kreuter eta/., 1992). Further experiments, however, are needed to prove whether the observed exudation of aeroplysinin-1 (100 g of sponge was reported to release a total of 13 mg of aeroplysininl over a 10 day period) (Kreuter et al, 1992) is sufficient to suppress the growth of potential epibionts or pathogenic microorganisms.

The polyphagous marine snail L. littorea was used as a model to study the repellent properties of the defense metabolites from ~ aerophoba against molluscs. Aeroplysinin-1 (3) and dienone (4) clearly elicited avoidance behaviour of L./ittorea. The EC50 of compound 3 which was most active was observed at 0.1 mM (Fig. 6). In comparison, the total concentrations of brominated alkaloids in V. aerophoba are known to vary from 10 to 15 mM (Teeyapant et aZ, 1993a). Whereas aeroplysinin-1


(3) or dienone (4) showed no acute toxicity towards L. littorea, preliminary experiments revealed pronounced larval mortality (70-80% larval mortality compared to controls) of the marine fish Blennius pavo Risso (Blenniidae) when exposed to aeroplysinin-1 (3) or dienone (4) at concentrations of 0.1 mM each, whereas aerophobin-2 (2) proved again to be inactive (Weiss et al., unpublished results). The latter preliminary data indicate that the defensive nature of aeroplysinin- 1 (3) and dienone (4) may extend to even more targets (e.g. marine fish) than analysed in the present study.

Acknowledgements- -F inanc ia l support from the Deutsche Forschu ngsgemeinschaft ("Sonderforschungsbereich 251 der Universit~t W0rzburg") and from the "'Fonds der Chemischen lndustrie" (both to P. Proksch) is gratefully acknowledged. R. Ebel wishes to thank the "Dr Hilmer- Stiftung im Stifterverband f0r die Deutsche Wissenschaft'" for a scholarship. Furthermore we are indebted to Professor W. E. G. M011er (Univ. Mainz, Germany) for a sample of V. aerophoba and to Dr H. v. Westernhagen (BAH, Germany) for giving us the opportunity to perform experiments with B. pavo.

R e f e r e n c e s Bakus, G. J., Targett, N. M. and Schulte, B. (1986) Chemical ecology of marine organisms: An overview.

J. Chem. EcoL 12, 951-987. Braekman, J. C. and Daloze, D. (1986) Chemical defense in sponges. Pure Appl. Chem. 58, 357-364. Bulich, A. A,, Ker-Kong, T. and Scheibner, G. (1990) The luminescent bacteria toxicity test: Its potential as

an in vitro alternative. J. Bioluminesc. Chemoluminesc. 5, 71-77. Burkholder, P. R. (1973) Ecology of marine antibiotics and coral reefs. In: Biology and Geology of Coral

Reefs (Jones, O. A. and Endean, R.), pp. 117-182. Academic Press, New York. Davis, A. R. (1991 ) Alkaloids and ascidian chemical defense: Evidence for the ecological role of natural

products from Eudistoma olivaceum. Mar. Biol. 111,375-379. Davis, A. R., Targett, N. M., McConnell, O. J. and Young, C. M. (1989) Epibiosis of marine algae and

benthic invertebrates: Natural products chemistry and other mechanisms inhibiting settlement and overgrowth. In: Bioorganic Marine Chemistry, Vol. 3 (Scheuer, P. J., ed,), pp. 85-114. Springer-Verlag, Berlin.

Hambsch, B., Pelouin, C. and Werner, P. (1990) Der Leuchtbakterienhemmtest als bewertender Summenparameter for raffineriespezifische Schadstoffe. Vom Wasser 74, 65-74.

Krebs, F. (1983) Toxizit~itstests mit gefriergetrockneten Leuchtbakterien. GWA 63, 173-230. Kreuter, M. H., Robitzki, A., Chang, S., Steffen, R., Michaelis, M., Klajic, Z., Bachmann, M., Schroder, H. C.

and MOiler, W. E. G. (1992) Production of the cytostatic agent aeroplysinin by the sponge Verongia aerophoba in in vitro culture. Comp. Biochem. Physiol. 101 C, 183-187,

Lawton, L. A., Campbell, D. L., Beattie, K. A. and Codd, G. A. (1990) Use of a rapid bioluminescence assay for detecting cyanobacterial microcystin toxicity. Lett. Appl. Microbiol. 11,205-207.

Luckner, M. (1984) Secondary Metabolism in Microorganisms, Plants and Animals. Springer-Verlag, Berlin. McCaffrey, E. J. and Endean, R. (1985) Antimicrobial activity of tropical and subtropical sponges. Mar.

Biol. 89, 1 4 . Mebs, D. (1989) Gifte im Rift. Wissenschaftliche Verlagsgesellschaft, Stuttgart. Melton, T. and Bodnar, J. W. (1988) Molecular biology of marine microorganisms: Biotechnological

approaches to naval problems. Nav. Res. Rev. 40, 24-39. Teeyapant, R. (1994) Brominated secondary metabolites of the marine sponge Verongia aerophoba

Schmidt and the sponge feeding gastropod Tylodina perversa Gmelin: Identification, biological activities and biotransformation. Ph.D. thesis, Univ. W0rzburg.

Teeyapant, R., Kreis, P., Wray, V., Witte, L. and Proksch, P. (1993a) Brominated secondary compounds from the marine sponge Verongia aerophoba and the sponge feeding gastropod Tylodina perversa. Z Natudorsch. 48c, 640-644.

Teeyapant, R. and Proksch, P. (1993) Biotransformation of brominated compounds in the marine sponge Verongia aerophoba~evidence for an induced chemical defense? Naturwissenschaften 80, 369-370.

Teeyapant, R., Woerdenbag, H. J., Kreis, P., Hacker, J., Wray, V., Witte, L. and Proksch, P (1993b) Antibiotic and cytotoxic activity of brominated compounds from the marine sponge Verongia aerophoba. Z. Naturforsch. 48c, 939-945.

Tymiak, A. A. and Rinehart, K. L. (1981) Biosynthesis of dibromotyrosine-derived antimicrobial compounds by the marine sponge Aplysina fistularis (Verongia aurea). J. Am. Chem. Soc. 103, 6763- 6765.

Walls, J. T., Blackman, A. J. and Ritz, D. A. (1991) Distribution of amathamide alkaloids within single colonies of the bryozoan Amathia wilsonL J. Chem. EcoL 17, 1871-1881.

12 B. WEISS ETAL.

Yates, P., McLachlan, F. N., Rac, I. D., Rosenberger, M., Szabo, A. G., Willis, C., Cara, M. P., Behrorouz, M., Lakshmikantham, M. V. and Zeiger, W. (1973) Halophytine. Novel type of indole alkaloid. J. Am. Chem. Soc. 95, 7842-7850.

ZoBell, C. E. (1946) Marine Microbiology. Chronica Botanica Co., Waltham, Massachusetts.